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Analysis of biologically active compounds using high performance separation methods
Adamusová, Hana ; Bosáková, Zuzana (advisor) ; Jelínek, Ivan (referee) ; Tůma, Petr (referee)
In the first part of this doctoral thesis, a new analytical HPLC-MS/MS method for monitoring of concentration changes of 17β-estradiol (βE2) during in vitro mouse sperm capacitation was developed. Capacitation was performed for three initial concentrations of βE2 (200, 20 and 2 μg/L). For all the concentrations a similar trend for the total unbound βE2 was observed. In general, the βE2 concentration decreased to reach its minimum and then increased again. The position of the minimum differed for the individual tested βE2 concentrations. Experimentally obtained results were subjected to the kinetic analysis. The curves fitted through the experimentally determined points displayed an autocatalytic character. For the agreement between the curves obtained by fitting through the experimental points and the theoretical calculated curves, it is necessary to assume that the first step is adsorption of βE2 onto the surface of the sperm controlled by Langmuir isotherm. The kinetic study was also used to study the effects of fluorides and aluminium fluoride complexes on the capacitation of mouse sperm. The experimental points were in very good agreement with the shape of the theoretical curves and this fact verifies the mechanism of the mouse sperm capacitation kinetics. In the second part of this work, two...
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Chiral separation of biologically active compounds by chromatography
Landl, David ; Kalíková, Květa (advisor) ; Kubíčková, Anna (referee)
- 4 - Abstract in English This diploma thesis is focused on the evaluation and comparison of the enantioselective potential of two columns CHIRAL ART Amylose-SA and CDShell-RSP using a set of 29 chiral drugs in high performance liquid chromatography. The separations of enantiomers were performed in three modes: reversed-phase, normal-phase, and polar- organic mode. The CHIRAL ART Amylose-SA column was tested in normal-phase mode, the CDShell-RSP column in reversed-phase and polar-organic modes. The CHIRAL ART Amylose-SA column contains amylose tris(3,5- dimethylphenylcarbamate) immobilized on 3 µm porous silica gel particles. The CDShell-RSP column contains a chiral selector hydroxypropyl-β-cyclodextrin, which is covalently bonded on 2.7 µm superficially porous particles. In the normal-phase mode, mobile phases composed of hexane and propane-2-ol were used. Furthermore, the effect of various additives (triethylamine, diethylamine, trifluoroacetic acid, and the mixture of diethylamine and trifluoroacetic acid) in the mobile phase on the enantioseparation of chiral drugs was tested. The most universal additive was a mixture of diethylamine and trifluoroacetic acid. A total of 22 chiral drugs were enantioseparated on the CHIRAL ART Amylose-SA column, 10 of them were baseline separated. Mobile phases for...
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The application of high-efficiency separation methods for chiral and achiral separations
Šubová, Martina ; Bosáková, Zuzana (advisor) ; Jelínek, Ivan (referee) ; Fischer, Jan (referee)
In the first part of the doctoral thesis, the capillary electrophoresis was used to test the potential chiral separation properties of monosubstituted cyclodextrin derivatives, namely PEMEDA- and PEMPDA-β-cyclodextrins for the group of selected analytes. Both selectors exhibited excellent enantioseparation properties for N-boc-D,L-tryptophan, where the enantiomers were completely separated even at 0.5 mmol·l-1 concentration of the cyclodextrin derivative in the background electrolyte. However, the differences between the enantiodiscrimination properties of individual derivatives were minimal. The second test group consisted of two cyclodextrin derivatives, namely 2-O- and 3-O- cinnamyl-α-cyclodextrins. These derivatives are able to form supramolecular polymers in aqueous solutions that disintegrate at elevated temperature. The formation of these polymers was tested by NMR and DLS experiments. None of the tested cyclodextrin derivatives showed enantiodiscrimination properties towards a group of selected analytes. In the frame of antipredatory study, HPLC-MS/MS method working in HILIC mode was used for separation of ten pterin derivatives and riboflavin, which can be present as pigments in insects, reptiles or amphibians as a part of their warning coloration. The developed methodology was applied for...
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Chiral separation of diquats and determination of stability constants of their complexes with cyclodextrins by capillary electrophoresis
Bílek, Jan ; Kašička, Václav (advisor) ; Jelínek, Ivan (referee)
Capillary zone electrophoresis was used for chiral separation of eleven diquat derivatives. These N-heteroaromatic dications containing structural motif of 2,2'-bipyridine have recently been studied for their interesting electrochemical properties as well as for the axial chirality of their molecules. The combination of these properties could potencially lead to interesting applications in the future. For enantioseparation of diquats (DQ) commercially available randomly sulfated α-, β-, and γ-cyclodextrins with high degree of substitution were used. A succesfull chiral separation was achieved using all of the three sulfated cyclodextrins as chiral selectors (CS). Baseline enantioseparation was achieved for 82 %, 91 % respectively 100 % of the analyzed DQ in the presence of HS-α-CD, HS-β-CD, HS-γ-CD respectively. The highest separation efficiency and resolution were obtained in the backround electrolyte containing 22 mmol/L NaOH, 35 mmol/L H3PO4 (pH2,5) and 6 mmol/L HS-β-CD. Using three available nonracemic DQ an identification of the particular M- and P-enantiomers was done for the three corresponding DQ structures. Apparent stabillity constants of complexes of the DQ derivatives with above mentioned cyclodextrins as CS were determined by means of capillary affinity electrophoresis. The stability...
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Characterization of new chiral separation systems for HPLC
Vozka, Jiří ; Tesařová, Eva (advisor) ; Sýkora, David (referee) ; Petr, Jan (referee)
The dissertation thesis is focused on the physico-chemical characterization of interaction mechanisms of chiral stationary phases based on derivatized cyclofructans. Correct interpretation of retention and enantiodiscrimination processes substantially facilitates the development and optimization of new enantioselective methods using cyclofructan-based chiral stationary phases. At first, the interaction mechanisms of three commercially available cyclofructan-based stationary phases were studied in normal-phase mode of liquid chromatography. Namely, systems using chiral stationary phases based on dimethylphenyl carbamate cyclofructan 7, R-naphtylethyl carbamate cyclofructan 6 and isopropyl carbamate cyclofructan 6 were studied. Linear free energy relationship model was used as a basic tool for characterization of interactions on the stationary phases. The mentioned model revealed that the main interactions contributing to retention in cyclofructan-based systems are hydrogen bond acidity and dipolarity/polarizibility, while dispersion interactions cause decrease of retention. The impact of oligosaccharide skeleton of the cyclofructan selector on the enantioselectivity was elucidated by the comparison with seemingly analogous cyclodextrin-based chiral stationary phases. Cyclofructan-based chiral...
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Optimization of chromatographic parameters for chiral separation of biologically active compounds
Novák, Martin ; Kučera, Radim (advisor) ; Nobilis, Milan (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of: Pharmaceutical Chemistry and Pharmaceutical Analysis Student: Bc. Martin Novák Supervisor: doc. PharmDr. Radim Kučera, Ph.D. Consultant: Mgr. et Mgr. Rafael Doležal, Ph.D. Title of diploma thesis: Optimization of chromatographic parameters for chiral separation of biologically active compounds. The diploma thesis was focused on the development of a HPLC-UV method for the determination of K 1277 enantiomers of systematic name N-(2-((6-chloro-1,2,3,4-tetra- hydroacridin-9-yl)amino)hexyl-2-amino-3-(1H-indole-3-yl) propylamide dihydrochloride, which is one of the compounds from the tacrine-tryptophan hybrids group. These tacrine-tryptophan hybrids could be considered as promising candidates of potential drugs against Alzheimer's disease. The thesis brings an explanation of basic characteristic of chiral molecules, principles of chiral separation, pathophysiology, clinical manifestation and treatment of Alzheimer's disease and short characteristic of tacrine-tryptophan hybrids in the theoretical section. The aim of my diploma thesis was to find the optimal chromatographic conditions for separation of K 1277 enantiomers synthesized from tacrine and tryptophan fragments. The experimental part deals with the development of the chiral...
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The uset of HPLC in the field of chiral separations VI.
Marvalová, Jana ; Kučera, Radim (advisor) ; Pilařová, Pavla (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department: Department of Pharmaceutical chemistry and Pharmaceutical analysis Candidate: Jana Marvalová Supervisor: doc. PharmDr. Radim Kučera, PhD. Title of Thesis: The employment of HPLC in field of chiral separations VI. Boron clusters are synthetically prepared substances that are being intensively studied in connection with Boron Neutron Capture Therapy, the substitution of phenyl rings of molecules of already known drugs and as potential inhibitors of HIV protease. Boron clusters are symmetric molecules, however, by endo- or exo-skeletal substitution, the symmetry of the cluster is disrupted and enantiomers are formed. This diploma thesis is focused on the investigation of chromatographic conditions for chiral separations of cosanes (bis(dicarbollides) and 7,8-dicarb-nido-undecaborates derivatives using high performance liquid chromatography and chiral selectors based on cellulose and amylose in reverse-phase liquid chromatography. For this purpose, columns Lux Cellulose-1 and Lux Amylose-1 were selected with chiral selectors cellulose tris(3,5-dimethylphenylcarbamate) and amylose tris(3,5-dimethylphenylcarbamate), respectively. The mobile phases were mixtures of methanol or acetonitrile with sodium perchlorate or sodium chloride...
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Characterization of new chiral separation systems for HPLC
Vozka, Jiří
The dissertation thesis is focused on the physico-chemical characterization of interaction mechanisms of chiral stationary phases based on derivatized cyclofructans. Correct interpretation of retention and enantiodiscrimination processes substantially facilitates the development and optimization of new enantioselective methods using cyclofructan-based chiral stationary phases. At first, the interaction mechanisms of three commercially available cyclofructan-based stationary phases were studied in normal-phase mode of liquid chromatography. Namely, systems using chiral stationary phases based on dimethylphenyl carbamate cyclofructan 7, R-naphtylethyl carbamate cyclofructan 6 and isopropyl carbamate cyclofructan 6 were studied. Linear free energy relationship model was used as a basic tool for characterization of interactions on the stationary phases. The mentioned model revealed that the main interactions contributing to retention in cyclofructan-based systems are hydrogen bond acidity and dipolarity/polarizibility, while dispersion interactions cause decrease of retention. The impact of oligosaccharide skeleton of the cyclofructan selector on the enantioselectivity was elucidated by the comparison with seemingly analogous cyclodextrin-based chiral stationary phases. Cyclofructan-based chiral...
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The application of high-efficiency separation methods for chiral and achiral separations
Šubová, Martina
In the first part of the doctoral thesis, the capillary electrophoresis was used to test the potential chiral separation properties of monosubstituted cyclodextrin derivatives, namely PEMEDA- and PEMPDA-β-cyclodextrins for the group of selected analytes. Both selectors exhibited excellent enantioseparation properties for N-boc-D,L-tryptophan, where the enantiomers were completely separated even at 0.5 mmol·l-1 concentration of the cyclodextrin derivative in the background electrolyte. However, the differences between the enantiodiscrimination properties of individual derivatives were minimal. The second test group consisted of two cyclodextrin derivatives, namely 2-O- and 3-O- cinnamyl-α-cyclodextrins. These derivatives are able to form supramolecular polymers in aqueous solutions that disintegrate at elevated temperature. The formation of these polymers was tested by NMR and DLS experiments. None of the tested cyclodextrin derivatives showed enantiodiscrimination properties towards a group of selected analytes. In the frame of antipredatory study, HPLC-MS/MS method working in HILIC mode was used for separation of ten pterin derivatives and riboflavin, which can be present as pigments in insects, reptiles or amphibians as a part of their warning coloration. The developed methodology was applied for...
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The use of HPLC in the field of chiral separations V.
Burda, Jakub ; Kučera, Radim (advisor) ; Kastner, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department: Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Jakub Burda Supervisor: doc. PharmDr. Radim Kučera, Ph.D. Title of Thesis: The employment of HPLC in the field of chiral separations V. Continuously growing requirements of regulatory authorities for the quality and safety of medicines put a high pressure on manufacturers. Last decades are marked by optically pure drugs, whose developement goes hand in hand with developement of chiral syntheses and separations. The most used method in the field of separations are direct separations using chiral stationary phases. The most often used carrier for these stationary phases is silica, which may contain metal impurities on its surface, negatively impacting separation process. The focus of this thesis was testing of influuence of chromatographic conditions on chiral and achiral interactions of seven selected analytes with stationary phase. Column with native β-cyclodextrin as chiral selector was used for the testing. 1
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